The human blood protein Von Willebrand Factor (VWF) plays a critical role during thrombotic and hemostatic processes by forming a molecular bridge between extra-cellular matrix proteins exposed on the denuded blood vessel wall and platelets in the flow stream. Such platelet recruitment on the injured vessel wall contributes to plug and emboli formation in the vasculature. Several aspects of VWF function are regulated by fluid or hydrodynamic shear: i) The constitutively active blood metalloprotease ADAMTS-13 cleaves VWF, with proteolysis rate being tightly regulated by fluid shear. ii) VWF binding to GpIba on platelet surface is augmented by fluid shear, and platelet recruitment at sites of vascular injury also occurs in a shear-dependent manner. iii) In addition to ADAMTS-13, fluid shear promotes the self-association of VWF and this is an additional mechanism regulating VWF size in circulation. Since multiple functions of VWF are regulated by similar magnitudes of applied hydrodynamic forces, we suggest that these functions are regulated by common/overlapping structural changes. These changes likely occur in the globular head section of VWF that contains the D'D3, A1, A2 and A3 domains of the protein. In particular, our specific aims determine: 1) if the masking of the VWF-A1 domain by VWF-D'D3 contributes to reduced cell adhesion in the native protein, with fluid shear unmasking this molecular interaction. 2) if the binding of ADAMTS-13 to VWF changes the conformation of the A2-domain and if this acts in synergy with fluid shear to regulate proteolysis kinetics. 3) if VWF self-association precedes and enhances the rate of shear driven VWF-A2 proteolysis, and if this protein aggregation process also enhances the avidity of VWF-GpIb1 binding under shear. To address these aims, a series of single-domain, dual-domain and multimeric-VWF constructs are produced in mammalian expression systems. Panels of novel single-domain and multimeric-VWF FRET proteins are also made. Functional/structural studies are carried out to measure VWF binding, protein conformation change, platelet adhesion and activation using both flow cytometry and fluorescence/confocal microscopy. Surface plasmon resonance (SPR) provides measures of molecular binding affinity/kinetics. Tandem mass spectrometry is applied to elucidate structural changes promoted by shear. In terms of a bridge between these different experimental modalities, hydrodynamic modeling is applied to estimate the magnitude and nature of force applied under the variety of fluid shear conditions. In order to confirm the physiological relevance of the work, particular emphasis is placed on validating the proposed hypotheses in the milieu of whole human blood, and in the presence of physiological/pathological shear stress. Some hypotheses are also validated in a mouse model of arterial thrombosis. Together, the studies are designed to provide fundamental insight on the role of fluid shear in regulating VWF structure, size and function. Success in this application may spur additional investigations on molecular interactions in circulation, besides VWF, that are conditioned by flowing blood.